Hepatitis C virus NS2 protein inhibits DNA damage pathway by sequestering p53 to the cytoplasm

Abstract

Chronic hepatitis C virus (HCV) infection is an important cause of morbidity and mortality globally, and often leads to end-stage liver disease. The DNA damage checkpoint pathway induces cell cycle arrest for repairing DNA in response to DNA damage. HCV infection has been involved in this pathway. In this study, we assess the effects of HCV NS2 on DNA damage checkpoint pathway. We have observed that HCV NS2 induces ataxia-telangiectasia mutated checkpoint pathway by inducing Chk2, however, fails to activate the subsequent downstream pathway. Further study suggested that p53 is retained in the cytoplasm of HCV NS2 expressing cells, and p21 expression is not enhanced. We further observed that HCV NS2 expressing cells induce cyclin E expression and promote cell growth. Together these results suggested that HCV NS2 inhibits DNA damage response by altering the localization of p53, and may play a role in the pathogenesis of HCV infection.

Figure 1. Generation of stable cell lines expressing HCV NS2 protein.

(A) HepG2 cells are stably transfected with HCV-NS2 or HCV-FL. Immunofluorescence images displayed the expression of HCV NS2 (green color) in stably transfected HepG2 cells with HCV NS2 and HCV-FL. The nuclei (blue color) were stained with DAPI (4′, 6′-diamidino-2-phenylindole). Merged photographs are shown in right panel. (B) Cell lysates were prepared from HepG2 cells stably expressing HCV NS2 and HCV-FL. Protein expression of NS2 were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load.

Figure 2. HCV NS2 induces nuclear foci formation of phosphorylated Chk2.

(A) Confocal microscopy was performed for Chk2 localization in HepG2 cells stably expressing vector control or HCV NS2. HepG2 cells were treated with 15 µM cisplatin for 6 hrs and used as a positive control. Chk2 was stained with specific monoclonal antibody (green color) and nuclei were stained with DAPI (blue color). Merged photographs were shown in right panel. (B) Confocal microscopy was performed for phospho-Chk2 (Thr 68) localization in vector control and cisplatin treated HepG2 cells or HepG2 cells transfected with HCV NS2. Immunofluorescence images displayed the expression of HCV NS2 (green color) and phospho-Chk2 (red color) in control, cisplatin treated and HCV NS2 transfected HepG2 cells. The nuclei (blue color) were stained with DAPI. Merged photographs (right panel) were superimposed digitally for fine comparison.

Figure 3. HCV NS2 activates Chk2 in hepatocytes.

(A) Cell lysates were prepared from HepG2 cells stably expressing HCV NS2 and HCV NS5A. Protein expression of phospho-Chk2 (Thr-68), total Chk2, HCV proteins were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load. (B) Cell lysates were prepared from HepG2 cells stably transfected with vector control or HCV-FL. Protein expression of phospho-Chk2, and total Chk2 were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load. (C) Cell lysates were analyzed from mock or 15 µM cisplatin treated HepG2 cells, and protein expression of phospho-Chk2 and total Chk2 were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load. (D) Cell lysates were prepared from HepG2 cells expressing HCV NS2 and HCV-FL. Protein expression of phospho-H2AX (Ser-139) was analyzed by Western blot using specific antibody. UV treated cell lysates were used as positive control. The blot was reprobed with actin for a comparison of protein load.

Figure 4. HCV NS2 does not activate p53 or its downstream molecule.

(A) p53 expression was examined in HepG2 cells stably expressing HCV NS2 or HCV-FL and compared with control vector transfected cells using monoclonal antibody to p53. The blot was reprobed with an antibody to actin for comparison of equal protein load. (B) Immunofluorescence staining using a polyclonal antibody to p53 exhibits nuclear localization of p53 (red color) in mock-transfected cells (top row) or perinuclear localization of p53 in HCV NS2 (middle row) or HCV-FL (bottom row) transfected cells. HCV NS2 was stained with a monoclonal antibody (green color). The nuclei were stained with DAPI (blue color). Fluorescence images of left and middle panels were superimposed digitally for fine comparison (right panel). (C) p21 expression was examined in HepG2 cells stably expressing HCV NS2 or HCV-FL and compared with control vector transfected cells using monoclonal antibody to p21. The blot was reprobed with actin for comparison of equal protein load.

Figure 5. HCV NS2 promotes cellular proliferation.

(A) Cell proliferation was measured in control, HCV NS2 and HCV-FL stably transfected HepG2 cells by trypan blue exclusion method. The number of viable cells was counted at day 2 and day 4. The results are presented as means of three different experiments with standard errors. (B) Cell lysates prepared from HCV NS2 or HCV-FL stably transfected HepG2 cells were analyzed by western blot for the expression of cyclin E and p27 with respect to control vector transfected cells using specific antibody. The blot was reprobed with actin for comparison of equal protein load.